Antibodies are characterized by their specificity to identify antigens and by their binding properties. The development of monoclonal techniques has contributed to significant improvements in protein-chemical analyses of antibodies and applied technologies.
Although thermodynamic analysis and the measurement of binding reactions between antibodies and antigens can be carried out simply now, there is no detailed information on conformational changes in the antibody that accompany binding to the antigen. This is due to the difficulty of applying X-ray crystallographic analysis, which is widely used to analyze the three-dimensional protein structure, to huge proteins such as antibodies. Many structural analyses of antigen-antibody complexes are performed using Fab fragments of antibodies obtained by enzymatic digestion (Non-Patent Document 1). These analyses show that antigen binding induces conformational changes of several angstroms in amino acid residues in the antigen-binding site; however, it cannot be determined from the structural analysis of the fragments whether the changes are transmitted to a constant domain that is spatially distant from the binding site. On the other hand, antigen-antibody complexes have effector functions such as complement cascade activation, which does not exist in free antibodies. This suggests that the conformational change in the antibody caused by antigen binding is induced by the constant domain, to which the complement binds. It has, however, been interpreted that the effector functions of the complex are caused by aggregation since complement cascades are also activated by aggregates of antibodies. Meanwhile, Azuma et al. carefully analyzed the binding reaction of protein A, which is derived from Staphylococcus aureus, to antibodies, and found that antibodies bound to antigens and other antibodies unbound to antigens exhibited different reactivities to the protein A (Non-Patent Documents 2 and 3). However, the structural difference between free antibodies and antigen-antibody complexes, detected by protein A, was too small to be detected by ELISA etc.
Non-Patent Document 1: “Structural evidence for induced fit as a mechanism for antibody-antigen recognition”, James M. Rini, Ursula Schulze-Gahmen, and Ian A. Wilson, Science, 255: 959-965 (1992)
Non-Patent Document 2: “Evidence of allosteric conformational changes in the antibody constant region upon antigen binding”, Masayuki Oda, Haruo Kozono, Hisayuki Mori, and Takachika Azuma, International Immunology, 15: 417-426 (2003)
Non-Patent Document 3: “Conformational changes in the antibody constant domains upon hapten-binding”, Takuma Sagawa, Masayuki Oda, Misayuki Morii, Hisao Takizawa, Haruo Kozono, and Takachika Azuma, Molecular Immunology, 42: 9-18 (2005)